Biomarkers and methods for predicting benefit of adjuvant chemotherapy

ABSTRACT

Biomarkers, methods, assays, and kits are provided for predicting the efficacy of adjuvant chemotherapy (ACT) in a subject with early-stage non-small cell lung cancer (NSCLC).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.61/759,763, filed Feb. 1, 2013, which is hereby incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under AgreementCA119997, CA129343, CA163068, and CA118809 awarded by the NationalInstitutes of Health. The Government has certain rights in theinvention.

BACKGROUND

Lung cancer accounts for over 160,000 deaths per year in the U.S., morethan breast, colon, prostate and pancreatic cancer combined. The overallfive-year survival rate for lung cancer is approximately 15%, and unlikeother solid tumors, such as colon or breast cancer, little progress hasbeen made in improving survival. Early-stage non-small cell lung cancer(NSCLC) is primarily treated by surgical resection. Unfortunately, afterresection, one-third to one-half of early-stage patients will die ofmetastatic recurrence. Adjuvant chemotherapy (ACT) improves the survivalof patients with early-stage disease and has become the standardtreatment for patients with resected stage II-III NSCLC. However, thefive-year survival advantage of ACT is only 4%-15% suggesting that manypatients do not benefit. Management of early stage lung cancer followingsurgical resection still relies on metrics such as tumor size and lymphnode status to guide decision making regarding adjuvant chemotherapy(ACT). Given the morbidity associated with ACT, it is imperative todevelop new prognostic tools to identify those patients with highprobability of relapse.

SUMMARY

Biomarkers, methods, assays, and kits are provided for predicting thesurvival of a subject with a cancer, such as early-stage non-small celllung cancer (NSCLC). These biomarkers, methods, assays, and kits cantherefore be used to predict the benefit of adjuvant chemotherapy (ACT)for a subject based on their expected survivability. In someembodiments, the biomarkers, methods, assays, and kits also predict theefficacy of ACT in the subject. The assays and kits can contain primers,probes, or binding agents for detecting expression at least 2, 10, 20,30, 40, 50, 60, 70, 71, 72, 73, 74, or 75 of the genes listed in Table1.

The disclosed method can involve obtaining a biological sample from thesubject; determining levels of at least 2, 10, 20, 30, 40, 50, 60, 70,71, 72, 73, 74, or 75 genes listed in Table 1 in the biological sample.The method can further involve comparing the gene expression levels tocontrol values to produce a gene profile. The method can then comprisecalculating an E2F signature score from the gene profile. For example,in some embodiments, a high E2F signature score is an indication thatthe subject will benefit from ACT.

In particular, the biological sample can be RNA derived from formalinfixed paraffin embedded tissue. These slides are routinely collected forhistology and can be used as source of RNA to derive an E2F signaturescore. The method can further involve treating the subject with ACT ifthey have a high E2F signature score.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is an image of a Western blot for E2F1, E2F3A, E2F3B, E2F3A+B,E2F4, Rb proteins in H1299 and A549 NSCLC lines treated with siRNAstargeting Rb, E2F1, E2F3A, E2F3B, E2F4, or Actin.

FIG. 2 is a graph showing predictive effect (survival probability as afunction of time) of the E2F signature in a 133 patient cohort (JBR.10trial): interaction effect (HR=0.29; p=0.02). MR.L: Low MR; MR.H: HighMR; ACT: group with ACT; OBS: group without ACT.

DETAILED DESCRIPTION

Given the morbidity associated with ACT, it is imperative to develop newprognostic tools to identify those patients with a high probability ofrelapse. Toward this end, small inhibitory RNAs targeting multiple E2Fpathway components were used to derive an E2F gene expression signaturein vitro. This signature was refined by filtering for its componentsthat were altered in non-small cell lung cancers compared to normaltissue. Principle component analysis (PCA) was then used to identify asignature which was tested for correlation to overall survival in twolarge cohorts. The first of the two cohorts was the MolecularClassification of Lung Adenocarcinoma (MCLA) from the Director'sChallenge Consortium and the second was a novel database on 444 lungadenocarcinomas treated as a part of Moffitt's Total Cancer CareNetwork.

Disclosed are methods for predicting the survival of a subject with acancer, such as early-stage non-small cell lung cancer (NSCLC). Thismethod can therefore be used to predict the benefit of adjuvantchemotherapy (ACT) for a subject based on their expected survivability.In some embodiments, the method also predicts the efficacy of ACT in thesubject. The method generally involves first obtaining a biologicalsample from the subject, such as RNA derived from a tumor biopsy. Geneexpression assays can then be conducted on the biological sample todetermine levels of at least 2, 10, 20, 30, 40, 50, 60, 70, 71, 72, 73,74, or 75 of the disclosed E2F signature genes. The method can furtherinvolve obtaining a dataset comprising the levels of each gene and theninputting the data into an analytical classification process that usesthe data to classify the biological sample with an E2F signature score.

E2F Signature

The disclosed E2F signature is strongly prognostic. Additionally, usingJBR. 10 data for patients who either did or did not receive ACT allowedthe determination that patients having a high E2F signature benefit fromACT (have increased overall survival), whereas patients with a low E2Fsignature do not. Overall, these results indicate that this approachcould be optimized in the clinical setting to distinguish patientslikely to benefit from ACT from those who will not.

The disclosed method involves obtaining a biological sample from thesubject; determining gene expression levels of at least 2, 10, 20, 30,40, 50, 60, 70, 71, 72, 73, 74, or 75 genes listed in Table 1 in thebiological sample. Exemplary weights for calculating E2F score areprovided in Table 1; however, routine multivariate analysis can be usedto determine alternative weights for the genes in this list, or a subsetthereof, by comparing gene expression data in patient cohorts asdescribed herein.

TABLE 1 E2F Signature Genes and Weights Weight to calculate Gene E2Fscore ABAT −0.06618 ABCC6 −0.08934 ACOX2 −0.06001 AK2 −0.04452 ANXA1−0.03847 ARHGDIB −0.10618 ARL14 0.013557 BDH2 −0.10153 BIRC5 0.205929BLM 0.1584 BUB1B 0.206879 C1orf112 0.159249 CCNE2 0.164075 CDC6 0.191873CDCA4 0.167051 CENPF 0.184186 CENPQ 0.126635 CHST11 0.072747 CKS1B0.175074 CPM −0.07967 CYP1B1 −0.02709 DHFR 0.092405 DOCK4 −0.07588 EVI5−0.03151 FN1 0.000309 GATA3 −0.00604 GBP2 0.011828 GINS1 0.195944 GINS20.176601 GINS4 0.115602 GLIPR1 −0.01129 HMMR 0.181811 IDS −0.05545 IMPA20.041727 ISG20 0.02988 KIAA0101 0.077396 KIF15 0.200847 KIF4A 0.205465KIF5C 0.040016 LAMC2 0.018655 LARP6 0.021552 LAT2 −0.05019 LMNB10.160433 MCM10 0.196864 MCM2 0.188674 MCM4 0.176091 MDFIC −0.03356 MYB0.070217 NFE2L3 0.015477 NRP1 −0.06153 PLAT −0.04571 PLAUR 0.042204 PLK10.188229 PLSCR4 −0.09501 PRMT3 0.090478 PTHLH 0.031723 PTP4A1 −0.02083QKI −0.03663 RAD51 0.179822 RAD51AP1 0.193873 RASGRP1 −0.06656 RRAS2−0.0149 SEC61A2 0.104516 SFXN1 0.102801 SLC16A1 0.10311 SLC1A1 −0.05329SNAP25 0.0556 SOX4 0.024096 ST3GAL5 −0.11571 STIL 0.177318 SYT1 0.062172TGFB1I1 −0.05406 TK1 0.166273 TMEM156 −0.00983 TMPO 0.121226

The biological sample may comprise any clinically relevant tissuesample, such as a tumor biopsy. The sample may be taken from a human,or, in a veterinary context, from non-human animals such as ruminants,horses, swine or sheep, or from domestic companion animals such asfelines and canines. Additionally, the samples may be from frozen orarchived formalin-fixed, paraffin-embedded (FFPE) tissue samples.

General methods for RNA extraction are well known in the art and aredisclosed in standard textbooks of molecular biology, including Ausubelet al., ed., Current Protocols in Molecular Biology, John Wiley & Sons,New York 1987-1999. Methods for RNA extraction from paraffin embeddedtissues are disclosed, for example, in Rupp and Locker, Lab Invest.56:A67, (1987); and De Andres et al. Biotechniques 18:42-44, (1995). Inparticular, RNA isolation can be performed using a purification kit, abuffer set and protease from commercial manufacturers, such as Qiagen(Valencia, Calif.), according to the manufacturer's instructions. Forexample, total RNA from cells in culture can be isolated using QiagenRNeasy mini-columns. Other commercially available RNA isolation kitsinclude MASTERPURE™ Complete DNA and RNA Purification Kit (Epicentre,Madison, Wis.) and Paraffin Block RNA Isolation Kit (Ambion, Austin,Tex.). Total RNA from tissue samples can be isolated, for example, usingRNA Stat-60 (Tel-Test, Friendswood, Tex.). Total RNA from FFPE can beisolated, for example, using High Pure FFPE RNA Microkit, Cat No.04823125001 (Roche Applied Science, Indianapolis, Ind.). RNA preparedfrom a tumor can be isolated, for example, by cesium chloride densitygradient centrifugation. Additionally, large numbers of tissue samplescan readily be processed using techniques well known to those of skillin the art, such as, for example, the single-step RNA isolation processof Chomczynski (U.S. Pat. No. 4,843,155).

Gene Expression Assays

Methods of “determining gene expression levels” include methods thatquantify levels of gene transcripts as well as methods that determinewhether a gene of interest is expressed at all. A measured expressionlevel may be expressed as any quantitative value, for example, afold-change in expression, up or down, relative to a control gene orrelative to the same gene in another sample, or a log ratio ofexpression, or any visual representation thereof, such as, for example,a “heatmap” where a color intensity is representative of the amount ofgene expression detected. Exemplary methods for detecting the level ofexpression of a gene include, but are not limited to, Northern blotting,dot or slot blots, reporter gene matrix, nuclease protection, RT-PCR,microarray profiling, differential display, 2D gel electrophoresis,SELDI-TOF, ICAT, enzyme assay, antibody assay, and MNAzyme-baseddetection methods. Optionally a gene whose level of expression is to bedetected may be amplified, for example by methods that may include oneor more of: polymerase chain reaction (PCR), strand displacementamplification (SDA), loop-mediated isothermal amplification (LAMP),rolling circle amplification (RCA), transcription-mediated amplification(TMA), self-sustained sequence replication (3SR), nucleic acid sequencebased amplification (NASBA), or reverse transcription polymerase chainreaction (RT-PCR).

A number of suitable high throughput formats exist for evaluatingexpression patterns and profiles of the disclosed biomarkers. Numeroustechnological platforms for performing high throughput expressionanalysis are known. Generally, such methods involve a logical orphysical array of the subject samples, the biomarkers, or both. Commonarray formats include both liquid and solid phase arrays. For example,assays employing liquid phase arrays, e.g., for hybridization of nucleicacids, binding of antibodies or other receptors to ligand, etc., can beperformed in multiwell or microtiter plates. Microtiter plates with 96,384 or 1536 wells are widely available, and even higher numbers ofwells, e.g., 3456 and 9600 can be used. In general, the choice ofmicrotiter plates is determined by the methods and equipment, e.g.,robotic handling and loading systems, used for sample preparation andanalysis. Exemplary systems include, e.g., xMAP® technology from Luminex(Austin, Tex.), the SECTOR® Imager with MULTI-ARRAY® and MULTI-SPOT®technologies from Meso Scale Discovery (Gaithersburg, Md.), the ORCA™system from Beckman-Coulter, Inc. (Fullerton, Calif.) and the ZYMATE™systems from Zymark Corporation (Hopkinton, Mass.), miRCURY LNA™microRNA Arrays (Exiqon, Woburn, Mass.).

Alternatively, a variety of solid phase arrays can favorably be employedto determine expression patterns in the context of the disclosedmethods, assays and kits. Exemplary formats include membrane or filterarrays (e.g., nitrocellulose, nylon), pin arrays, and bead arrays (e.g.,in a liquid “slurry”). Typically, probes corresponding to nucleic acidor protein reagents that specifically interact with (e.g., hybridize toor bind to) an expression product corresponding to a member of thecandidate library, are immobilized, for example by direct or indirectcross-linking, to the solid support. Essentially any solid supportcapable of withstanding the reagents and conditions necessary forperforming the particular expression assay can be utilized. For example,functionalized glass, silicon, silicon dioxide, modified silicon, any ofa variety of polymers, such as (poly)tetrafluoroethylene,(poly)vinylidenedifluoride, polystyrene, polycarbonate, or combinationsthereof can all serve as the substrate for a solid phase array.

In one embodiment, the array is a “chip” composed, e.g., of one of theabove-specified materials. Polynucleotide probes, e.g., RNA or DNA, suchas cDNA, synthetic oligonucleotides, and the like, or binding proteinssuch as antibodies or antigen-binding fragments or derivatives thereof,that specifically interact with expression products of individualcomponents of the candidate library are affixed to the chip in alogically ordered manner, i.e., in an array. In addition, any moleculewith a specific affinity for either the sense or anti-sense sequence ofthe marker nucleotide sequence (depending on the design of the samplelabeling), can be fixed to the array surface without loss of specificaffinity for the marker and can be obtained and produced for arrayproduction, for example, proteins that specifically recognize thespecific nucleic acid sequence of the marker, ribozymes, peptide nucleicacids (PNA), or other chemicals or molecules with specific affinity.

Microarray expression may be detected by scanning the microarray with avariety of laser or CCD-based scanners, and extracting features withnumerous software packages, for example, IMAGENE™ (Biodiscovery),Feature Extraction Software (Agilent), SCANLYZE™ (Stanford Univ.,Stanford, Calif.), GENEPIX™ (Axon Instruments).

In some embodiments, the nCounter® Analysis system (NanostringTechnologies, Seattle, Wash.) is used to detect intrinsic geneexpression. This system is described in International Patent ApplicationPublication No. WO 08/124,847 and U.S. Pat. No. 8,415,102, which areeach incorporated herein by reference in their entireties for theteaching of this system. The basis of the nCounter® Analysis system isthe unique code is assigned to each nucleic acid target to be assayed.The code is composed of an ordered series of colored fluorescent spotswhich create a unique barcode for each target to be assayed. A pair ofprobes is designed for each DNA or RNA target, a biotinylated captureprobe and a reporter probe carrying the fluorescent barcode. This systemis also referred to, herein, as the nanoreporter code system.

Specific reporter and capture probes are synthesized for each target.Briefly, sequence-specific DNA oligonucleotide probes are attached tocode-specific reporter molecules. Preferably, each sequence specificreporter probe comprises a target specific sequence capable ofhybridizing to no more than one gene of Table 1 and optionally comprisesat least two, at least three, or at least four label attachment regions,said attachment regions comprising one or more label monomers that emitlight. Capture probes are made by ligating a second sequence-specificDNA oligonucleotide for each target to a universal oligonucleotidecontaining biotin. Reporter and capture probes are all pooled into asingle hybridization mixture, the “probe library”. Preferably, the probelibrary comprises a probe pair (a capture probe and reporter) for eachof the genes in Table 1.

The relative abundance of each target is measured in a singlemultiplexed hybridization reaction. The method comprises contacting abiological sample with a probe library, the library comprising a probepair for the genes in Table 1, such that the presence of the target inthe sample creates a probe pair—target complex. The complex is thenpurified. More specifically, the sample is combined with the probelibrary, and hybridization occurs in solution. After hybridization, thetripartite hybridized complexes (probe pairs and target) are purified ina two-step procedure using magnetic beads linked to oligonucleotidescomplementary to universal sequences present on the capture and reporterprobes. This dual purification process allows the hybridization reactionto be driven to completion with a large excess of target-specificprobes, as they are ultimately removed, and, thus, do not interfere withbinding and imaging of the sample. All post hybridization steps arehandled robotically on a custom liquid-handling robot (Prep Station,NanoString Technologies).

Purified reactions are deposited by the Prep Station into individualflow cells of a sample cartridge, bound to a streptavidin-coated surfacevia the capture probe, electrophoresed to elongate the reporter probes,and immobilized. After processing, the sample cartridge is transferredto a fully automated imaging and data collection device (DigitalAnalyzer, NanoString Technologies). The expression level of a target ismeasured by imaging each sample and counting the number of times thecode for that target is detected. Data is output in simple spreadsheetformat listing the number of counts per target, per sample.

This system can be used along with nanoreporters. Additional disclosureregarding nanoreporters can be found in International Publication No. WO07/076,129 and WO 07/076,132, and US Patent Publication No. 2010/0015607and 2010/0261026, the contents of which are incorporated herein in theirentireties. Further, the term nucleic acid probes and nanoreporters caninclude the rationally designed (e.g. synthetic sequences) described inInternational Publication No. WO 2010/019826 and US Patent PublicationNo. 2010/0047924, incorporated herein by reference in its entirety.

Calculation of E2F Signature Score

From the disclosed gene expression values, a dataset can be generatedand inputted into an analytical classification process that uses thedata to classify the biological sample with an E2F signature score.

The data may be obtained via any technique that results in an individualreceiving data associated with a sample. For example, an individual mayobtain the dataset by generating the dataset himself by methods known tothose in the art. Alternatively, the dataset may be obtained byreceiving a dataset or one or more data values from another individualor entity. For example, a laboratory professional may generate certaindata values while another individual, such as a medical professional,may input all or part of the dataset into an analytic process togenerate the result.

Prior to input into the analytical process, the data in each dataset canbe collected by measuring the values for each marker, usually induplicate or triplicate or in multiple replicates. The data may bemanipulated, for example raw data may be transformed using standardcurves, and the average of replicate measurements used to calculate theaverage and standard deviation for each patient. These values may betransformed before being used in the models.

For example, it is often useful to pre-process gene expression data, forexample, by addressing missing data, translation, scaling,normalization, weighting, etc. Multivariate projection methods, such asprincipal component analysis (PCA) and partial least squares analysis(PLS), are so-called scaling sensitive methods. By using prior knowledgeand experience about the type of data studied, the quality of the dataprior to multivariate modeling can be enhanced by scaling and/orweighting. Adequate scaling and/or weighting can reveal important andinteresting variation hidden within the data, and therefore makesubsequent multivariate modeling more efficient. Scaling and weightingmay be used to place the data in the correct metric, based on knowledgeand experience of the studied system, and therefore reveal patternsalready inherently present in the data. For example, the weightsprovided in Table 1 can be used with the listed genes.

If possible, missing data, for example gaps in column values, should beavoided. However, if necessary, such missing data may replaced or“filled” with, for example, the mean value of a column (“mean fill”); arandom value (“random fill”); or a value based on a principal componentanalysis (“principal component fill”).

“Translation” of the descriptor coordinate axes can be useful. Examplesof such translation include normalization and mean centering.“Normalization” may be used to remove sample-to-sample variation. Somecommonly used methods for calculating normalization factor include: (i)global normalization that uses all genes on the array; (ii) housekeepinggenes normalization that uses constantly expressedhousekeeping/invariant genes; and (iii) internal controls normalizationthat uses known amount of exogenous control genes added duringhybridization. In some embodiments, the intrinsic genes disclosed hereincan be normalized to control housekeeping genes. It will be understoodby one of skill in the art that the methods disclosed herein are notbound by normalization to any particular housekeeping genes, and thatany suitable housekeeping gene(s) known in the art can be used.

Many normalization approaches are possible, and they can often beapplied at any of several points in the analysis. In one embodiment,data is normalized using the LOWESS method, which is a global locallyweighted scatter plot smoothing normalization function. In anotherembodiment, data is normalized to the geometric mean of set of multiplehousekeeping genes.

“Mean centering” may also be used to simplify interpretation. Usually,for each descriptor, the average value of that descriptor for allsamples is subtracted. In this way, the mean of a descriptor coincideswith the origin, and all descriptors are “centered” at zero. In “unitvariance scaling,” data can be scaled to equal variance. Usually, thevalue of each descriptor is scaled by 1/StDev, where StDev is thestandard deviation for that descriptor for all samples. “Pareto scaling”is, in some sense, intermediate between mean centering and unit variancescaling. In pareto scaling, the value of each descriptor is scaled by1/sqrt(StDev), where StDev is the standard deviation for that descriptorfor all samples. In this way, each descriptor has a variance numericallyequal to its initial standard deviation. The pareto scaling may beperformed, for example, on raw data or mean centered data.

“Logarithmic scaling” may be used to assist interpretation when datahave a positive is skew and/or when data spans a large range, e.g.,several orders of magnitude. Usually, for each descriptor, the value isreplaced by the logarithm of that value. In “equal range scaling,” eachdescriptor is divided by the range of that descriptor for all samples.In this way, all descriptors have the same range, that is, 1. However,this method is sensitive to presence of outlier points. In“autoscaling,” each data vector is mean centered and unit variancescaled. This technique is a very useful because each descriptor is thenweighted equally, and large and small values are treated with equalemphasis. This can be important for genes expressed at very low, butstill detectable, levels.

The methods described herein may be implemented and/or the resultsrecorded using any device capable of implementing the methods and/orrecording the results. Examples of devices that may be used include butare not limited to electronic computational devices, including computersof all types. When the methods described herein are implemented and/orrecorded in a computer, the computer program that may be used toconfigure the computer to carry out the steps of the methods may becontained in any computer readable medium capable of containing thecomputer program. Examples of computer readable medium that may be usedinclude but are not limited to diskettes, CD-ROMs, DVDs, ROM, RAM, andother memory and computer storage devices. The computer program that maybe used to configure the computer to carry out the steps of the methodsand/or record the results may also be provided over an electronicnetwork, for example, over the internet, an intranet, or other network.

This data can then be input into the analytical process with definedparameter. The analytic classification process may be any type oflearning algorithm with defined parameters, or in other words, apredictive model. In general, the analytical process will be in the formof a model generated by a statistical analytical method such as thosedescribed below. Examples of such analytical processes may include alinear algorithm, a quadratic algorithm, a polynomial algorithm, adecision tree algorithm, or a voting algorithm.

Using any suitable learning algorithm, an appropriate reference ortraining dataset can be used to determine the parameters of theanalytical process to be used for classification, i.e., develop apredictive model. The reference or training dataset to be used willdepend on the desired classification to be determined. The dataset mayinclude data from two, three, four or more classes.

The number of features that may be used by an analytical process toclassify a test subject with adequate certainty is 2 or more. In someembodiments, it is 3 or more, 4 or more, 10 or more, or between 10 and74. Depending on the degree of certainty sought, however, the number offeatures used in an analytical process can be more or less, but in allcases is at least 2. In one embodiment, the number of features that maybe used by an analytical process to classify a test subject is optimizedto allow a classification of a test subject with high certainty.

Suitable data analysis algorithms are known in the art. In oneembodiment, a data analysis algorithm of the disclosure comprisesClassification and Regression Tree (CART), Multiple Additive RegressionTree (MART), Prediction Analysis for Microarrays (PAM), or Random Forestanalysis. Such algorithms classify complex spectra from biologicalmaterials to distinguish subjects as normal or as possessing biomarkerlevels characteristic of a particular disease state. In otherembodiments, a data analysis algorithm of the disclosure comprises ANOVAand nonparametric equivalents, linear discriminant analysis, logisticregression analysis, nearest neighbor classifier analysis, neuralnetworks, principal component analysis, quadratic discriminant analysis,regression classifiers and support vector machines. While suchalgorithms may be used to construct an analytical process and/orincrease the speed and efficiency of the application of the analyticalprocess and to avoid investigator bias, one of ordinary skill in the artwill realize that computer-based algorithms are not required to carryout the methods of the present disclosure.

As will be appreciated by those of skill in the art, a number ofquantitative criteria can be used to communicate the performance of thecomparisons made between a test marker profile and reference markerprofiles. These include area under the curve (AUC), hazard ratio (HR),relative risk (RR), reclassification, positive predictive value (PPV),negative predictive value (NPV), accuracy, sensitivity and specificity,Net reclassification Index, Clinical Net reclassification Index. Inaddition, other constructs such a receiver operator curves (ROC) can beused to evaluate analytical process performance.

Predicting Cancer Survivability

The disclosed biomarkers, methods, assays, and kits can be used topredict the survivability of a subject with a cancer. The disclosedbiomarkers, methods, assays, and kits are particularly useful to predictsurvivability of early stage cancers where aggressive treatments are notroutinely used. For example, markers, methods, assays, and kits can beused to predict the survivability of a subject with early stagenon-small cell lung cancer (NSCLC). However, other cancers may benefitfrom these biomarkers, methods, assays, and kits to predict the benefitof aggressive treatment. For example, the cancer of the disclosedmethods can be any cell in a subject undergoing unregulated growth,invasion, or metastasis. In some aspects, the cancer can be any neoplasmor tumor for which radiotherapy is currently used. Alternatively, thecancer can be a neoplasm or tumor that is not sufficiently sensitive toradiotherapy using standard methods. Thus, the cancer can be a sarcoma,lymphoma, leukemia, carcinoma, blastoma, or germ cell tumor. Arepresentative but non-limiting list of cancers that the disclosedcompositions can be used to treat include lymphoma, B cell lymphoma, Tcell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloid leukemia,bladder cancer, brain cancer, nervous system cancer, head and neckcancer, squamous cell carcinoma of head and neck, kidney cancer, lungcancers such as small cell lung cancer and non-small cell lung cancer,neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer, prostatecancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas ofthe mouth, throat, larynx, and lung, colon cancer, cervical cancer,cervical carcinoma, breast cancer, epithelial cancer, renal cancer,genitourinary cancer, pulmonary cancer, esophageal carcinoma, head andneck carcinoma, large bowel cancer, hematopoietic cancers; testicularcancer; colon and rectal cancers, prostatic cancer, and pancreaticcancer.

Adjuvant Therapy

The calculated E2F signature score can be used to predict the benefit ofan adjuvant therapy for a subject based on their expected survivability.In some embodiments, the method also predicts the efficacy of adjuvanttherapy in the subject. Adjuvant therapy is additional treatment givenafter surgery to reduce the risk that the cancer will come back.Adjuvant treatment may include chemotherapy (the use of drugs to killcancer cells) and/or radiation therapy (the use of high energy x-rays tokill cancer cells).

As an example, the treatment for stage I, II, and IIIA NSCLC includessurgery to remove the tumor and the surrounding lung tissue and lymphnodes. The stage of NSCLC is described by a number, one through four(Roman numerals I-IV). A higher stage of cancer means that the risk thatthe cancer may come back is also higher. Stage I NSCLC means that thecancer has not spread to nearby lymph nodes. Stage IA means the primarytumor is relatively small. Stage IB means the primary tumor isrelatively large, or is located in a place where it is more likely tospread. A stage I cancer can usually be removed by surgery. Stage IINSCLC describes a cancer that may have spread to nearby lymph nodes.Stage IIA means the primary tumor is relatively small. Stage IIB meansthe primary tumor is relatively large, or is located in a place where itis more likely to spread. In a stage II cancer, both the tumor and theaffected lymph nodes can usually be removed by surgery. Stage III NSCLCmay be difficult to remove with surgery. When the cancer has spread tolymph nodes in the center of the chest, on the same side as where thecancer started, it is known as stage IIIA. When the cancer spreads tolymph nodes on the opposite side of the chest, it is known as stageIIIB. In general, surgery is not used for any stage IIIB lung cancer.Stage IV NSCLC has spread through the bloodstream to areas of the bodyoutside of the lung and is not treated with surgery.

The ASCO and CCO provide recommendations for adjuvant chemotherapy (ACT)treatment for stage I, II, and IIIA NSCLC. Chemotherapy after surgery toremove the lung cancer is recommended for patients with stage IIA, IIB,and IIIA NSCLC because clinical trials have shown that it may helppatients live longer. However, the five-year survival advantage of ACTis only 4%-15% suggesting that many patients do not benefit. Forexample, chemotherapy after surgery is generally not recommended forpatients with stage IA NSCLC because there is not enough evidence toshow that chemotherapy helps these patients live longer, and becausethese patients tend to have a good chance of long-term survival withsurgery alone. For the same reasons, chemotherapy for stage IB NSCLC isgenerally not recommended for every patient, but it may be appropriatein some situations. Moreover, the side effects of chemotherapy mayinclude fatigue, nausea and/or vomiting, appetite loss, and irritationaround the vein where the chemotherapy is injected. Other, less commonside effects include anemia (a decrease in the number of red bloodcells) fever with a low number of white blood cells, hair loss,constipation, peripheral neuropathy (a numbness, or tingling of thefingertips and/or toes), kidney damage, and hearing loss. Often, theseside effects go away after treatment, but damage to the nerves, kidneys,or hearing may be permanent. Because some patients (1%) who develop aninfection while their white blood count is low from chemotherapy havedied, it is desirable to avoid the morbidity associated with ACT if thesubject is not likely to benefit.

Radiation treatment after surgery is not generally recommended forpatients with stage IA, IB, IIA, or IIB NSCLC because clinical trialshave shown that it does not help patients live longer. Moreover, theside effects of radiation therapy may include difficulty breathing, asore throat, difficulty eating or swallowing, and fatigue.

The disclosed E2F signature score can be used to identify whether thesubject will have improve survivability if treated with ACT and may alsopredict benefit of radiation therapy. For example, the method caninvolve administering ACT and/or radiation therapy to the subject if ahigh E2F signature score is calculated. The method is particularlyuseful in early-stage cancers where adjuvant therapy is not routinelyprescribed. For example, in some embodiments, the subject has beendiagnosed with Stage I or Stage II NSCLC.

EXAMPLES Example 1 E2F Signature for Adjuvant Chemotherapy Survival

Results

The E2F/Rb pathway is central to the regulation of the mammalian cellcycle, and thus, it appears a reasonable target for the development ofchemotherapeutic agents (Ma, Y., et al., Cancer Res, 2008.68(15):6292-9) as well as potential prognostic or predictive marker fortumor progression (Sage, J., Nat Med, 2007. 13(1):30-1; La Thangue, N.B., Nat Cell Biol, 2003. 5(7):587-9; Johnson, D. G. and J. Degregori,Curr Mol Med, 2006. 6(7):731-8). Unfortunately, the E2F pathway can bealtered to varying degrees and by multiple molecular mechanisms, andthus, devising a straightforward clinical assay that would reflectdisruption of the E2F pathway as a whole and with a “singular”measurement has been elusive. To address this weakness, an siRNAapproach combined with microarray profiling was used to derive amRNA-based gene signature that reflects deactivation of the Rb pathway.

To accomplish this goal six siRNAs were developed that couldspecifically and efficiently deplete lung cancer cell lines ofindividual E2F components. Due to their biological prominence, E2F1,E2F3 A and B, E2F3 and Rb were chosen as targets. FIG. 1 demonstratesthe efficiency and specificity of these siRNAs. H1299 and A549 NSCLClines were treated with siRNAs targeting Rb, E2F1, E2F3A, E2F3B, E2F4,or Actin and then evaluated by Western blot for E2F1, E2F3A, E2F3B,E2F3A+B, E2F4, Rb protein expression.

Next, these two cell lines were subjected to microarray profiling todetect genes whose expression levels were significantly altered by thesedepletion studies. These lists of genes were then filtered to identifygenes that were altered in 5 of the six depletions and further filteredto identify a list of one-hundred genes. Principle component analysiswas then used to represent the signature which was tested forcorrelation to overall survival in two large cohorts. The first of thetwo cohorts was the Molecular Classification of Lung Adenocarcinoma(MCLA) from the Director's Challenge Consortium and the second was adatabase on 444 lung adenocarcinomas treated as a part of Moffitt'sTotal Cancer Care Network. The E2F signature is strongly prognostic inboth cohorts with P values of 3.52×10⁻⁷ and 3.11×10⁻⁷, respectively.

Additionally, using a published dataset for patients who either did ordid not receive ACT, it was possible to determine that patients having ahigh E2F signature benefit from ACT (have increased overall survival),whereas patients with a low E2F signature do not (FIG. 2). Overall,these results indicate that this approach could be optimized in theclinical setting to distinguish patients likely to benefit from ACT fromthose who will not.

Materials and Methods

Derivation of E2F Score:

An overall E2F score was generated using principal component analysis toreflect the combined effect of the E2F targeted genes. Specifically, thefirst principal component (a weighted average expression among the E2Fgenes), as it accounts for the largest variability in the data, was usedto represent the overall expression level for the signature. Thisapproach has been used to derive the malignancy-risk gene signature inlung and breast cancer study (Aberle, D. R., et al., N Engl J Med, 2011.365(5):395-409).

Evaluation of Predictive Feature:

For the predictive value, treatment effect (compared to control group)was evaluated to see any association with overall survival within eachsignature risk group (low- and high-score). In addition, an interactionmodel was conducted to determine any significance of the interactionterm (between the treatments and the signature). A significantinteraction effect could suggest differential treatment effects betweenthe signature risk groups. Two datasets were used for evaluation:Director's Challenge Consortium dataset and GSE 14814 dataset.

Principal component analysis was first implemented on the Director'sChallenge Consortium data to obtain the E2F score which was constructedbased on the loading coefficients from the first principal component.The same loading coefficients were also used to compute the E2F scorefor the GSE 14814 dataset. The median of the E2F score in the Director'sChallenge Consortium dataset was used as the cutoff to form low and highE2F score groups in each of the both datasets to test the predictiveeffect.

Example 2 NanoString™ Assay to Obtain E2F Signature

A NanoString™-based practical molecular assay was developed to analyzeRNA derived from formalin fixed paraffin embedded (FFPE) samples toidentify those early-staged NSCLC patients who are mostly likely tobenefit clinically from ACT. Thus far, Affymetrix®-based gene expressiondata has been used to define the E2F signature. Unfortunately,Affymetrix®-based assays require the isolation of large amounts of freshfrozen (FF) tissues which are generally not available for the majorityof archived patient sample since maintaining frozen samples is verycostly. In contrast, FFPE tissues are collected and stored long-term onall surgical patients and represent a vast reservoir of archival tumorspecimens. Recent studies demonstrate that mRNA sufficient in qualityand quantity can be retrieved from lung cancer FFPE tissues allowingrobust prediction of high risk lung cancer patients after surgicalresection similar to that found with fresh frozen tissue (Kratz, J. R.,et al., Lancet, 2012. 379(9818):823-32; Xie, Y., et al., Clin CancerRes, 2011 17(17):5705-14). A potential roadblock in the translation ofthese recent findings into clinical application is that neither usedassays that can be easily adapted to clinic. To address this weakness,the NanoString nCounter™ format can be used (Geiss, G. K., et al., NatBiotechnol, 2008. 26(3):317-25). The NanoString™ assay is direct (thereis no amplification steps that can bias signal strength), it has fewsteps and no enzymatic steps that might be inhibited by contaminants(Malkov, V. A., et al., BMC Res Notes, 2009. 2:80). The assay allowsnumerous probes in a single reaction (up to 800) and it is ideal forsmall nucleic acid fragments such as those present in formalin-fixedparaffin embedded tissues (Reis, P. P., et al., BMC Biotechnol, 2011.11:46). The NanoString™ assay has the same sensitivity as quantitativePCR methods (requiring only 100 ng of material) and demonstrates goodconcordance with these assays and microarray assays in directcomparisons (Reis, P. P., et al., BMC Biotechnol, 2011. 11:46; NorthcottP. A., Acta Neuropathol, 2012. 123(4):615-26; Barlin J. N., GynecolOncol. 2012).

A 75-gene signature was identified that can be used to determine ifpatients with early-stage NSCLC are likely to benefit from adjuvantchemotherapy (ACT) using NanoString™ analysis of RNA derived from FFPE.This gene signature was derived from a comprehensive analysis of theE2F/Rb pathway in vitro using siRNA, and has been found to correlatewith overall survival in two large cohorts. The first of the cohorts wasthe Molecular Classification of Lung Adenocarcinoma (MCLA) from theDirector's Challenge Consortium (p=3.52×10⁻⁷) and the second was a noveldatabase of 444 lung adenocarcinomas treated as a part of Moffitt'sTotal Cancer Care Program (p=3.11×10⁻⁷). The E2F signature is stronglyprognostic in both of these cohorts. Moreover, using a 133 patientcohort from the JBR. 10 trial (Zhu et al. Journal of Clinical Oncology(2010)28:4417), it was determined that patients with a high E2Fsignature benefit from ACT, whereas patients with a low E2F signature donot (p=0.01). The assay can be run using RNA extracted fromparaffin-embedded tissue samples and a validated NanoString® platformfor simple profiling of gene expression at reduced time and materialcosts.

This technology is an mRNA-based gene signature that reflectsdeactivation of the Rb pathway and is intended to objectively helpphysicians predict Stage Ib-II NSCLC patient response to adjuvantchemotherapy (ACT, cisplatin/vinorelbine).

FFPE and FF tissue was obtained from a cohort of 48 patients at Moffitt.Microarray and NanoString assays were performed on the fresh frozen andNanoString on the FF and FFPE RNA. A near perfect correlation wasobtained for NanoString assay of either FFPE tissue or FF tissue, andgood correlations were obtained with microarray data. This also resultedin a trimming of the codesets and controls.

Table 1 includes the current genes list (75 genes). Assays can alsoinclude internal controls (e.g., C2orf42, DEDD, GIGYF2, HDAC3, PRDM4,SART3, USP4, and BIRC6) that are expressed at very consistent level inseveral databases to allow for normalization between samples and batchesof samples using RNA extracted from paraffin-embedded tissue using avalidated NanoString® platform for simple profiling of gene expression.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A method for treating a subject with early-stagenon-small cell lung cancer (NSCLC), comprising a) measuring geneexpression levels with probes specific to a combination of 70 or moreE2F regulated genes selected from the group consisting of the geneslisted in Table 1 in a biological sample; b) comparing the geneexpression levels to control values to produce a gene profile; c)calculating an E2F signature score from the gene profile; and d)treating the subject with adjuvant chemotherapy (ACT) having an E2Fsignature score that is higher than a determined median cutoff level. 2.The method of claim 1, wherein the biological sample is RNA obtainedfrom formalin fixed paraffin embedded tissue.
 3. The method of claim 1,wherein the subject has been diagnosed with stage I NSCLC.
 4. The methodof claim 1, wherein gene expression levels of the genes listed in Table1 are determined using a nanoreporter code system.